Currently, there is no specific antiviral therapy available for the treatment of flavivirus infections, including West Nile Virus (WNV), a Biodefense Category B agent. Therefore, the development of novel classes of antiviral agents is of high priority for public health and defense against a possible bioterrorism attack. For this initiative, our goal is to develop small molecule inhibitors of WNV. In our preliminary experiments, we used a WNV replicon-based cell .culture high throughput screening platform to identify several candidate lead compounds and confirmed their inhibitory activity in WNV and other flavivirus infection assays in cell culture. Our collaborative group will bring the potential anti-viral lead compounds we discovered in our screens through a preclinical drug development program that includes lead optimization using structure-activity relationships (SAP) to enhance compound potency and drug-like properties. Preclinical toxicology and pharmacokinetic (PK) studies will be performed to help predict how the antiviral compounds distribute and behave in vivo and will include an analysis of cytotoxicity, solubility, metabolic stability, cell permeability, and plasma protein binding. As a cost-saving measure, we will perform preliminary preclinical toxicology studies in-house, for early identification of compounds that merit further consideration in advanced in vitro and In vivo toxicokinetics preclinical studies in small animals. Compounds with acceptable toxicity and PK profiles will be tested in efficacy studies using a well-characterized mouse model of WNV infection. The ability of our small molecule anti-WNV compounds to synergize with a neutralizing humanized monoclonal antibody to generate a more potent combination therapeutic regimen will also be studied. For the lead compounds with the greatest WNV anti-viral potential in vivo, virological studies will be performed to define the mechanism of action. It is expected that these studies will result in the production of an antiviral drug for future clinical trials to determine safety and efficacy against WNV, and possibly other flavivirus infections. Project Narrative There are currently no effective therapies against the Category B agent WNV to address the immediate health needs of the public in case of a bioterrorism incident. In this project, we are proposing to use the combined expertise of Apath.LLC and the Diamond laboratory at Washington University in St. Louis to develop anti-WNV compounds we discovered in our screening program into an antiviral drug to be tested in future clinical trials.